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MicroRNAs (miRNAs) are small non-coding RNAs that regulate mRNA expression mainly by silencing target transcripts via binding to miRNA recognition elements (MREs) in the 3’untranslated region (3’UTR). The identification of bona fide targets is challenging for researchers working on the functional aspect of miRNAs. Recently, we developed a method (miR-CATCH) based on biotinylated DNA antisense oligonucleotides that capture the mRNA of interest and facilitates the characterisation of miRNAs::mRNA interactions in a physiological cellular context. Here, the miR-CATCH technique was applied to the mesothelin (MSLN) gene and coupled with next generation sequencing (NGS), to identify miRNAs that regulate MSLN mRNA and that may be responsible for its increased protein levels found in malignant pleural mesothelioma (MPM). Biotinylated MSLN oligos were employed to isolate miRNA::MSLN mRNA complexes from a normal cell line (Met-5A) which expresses low levels of MSLN. MiRNAs targeting the MSLN mRNA were identified by NGS and miR-21-5p and miR-100-5p were selected for further validation analyses. MiR-21-5p was shown to be able to modulate MSLN expression in miRNA mimic experiments in a panel of malignant and non-malignant cell lines. Further miRNA inhibitor experiments and luciferase assays in Mero-14 cells validated miR-21-5p as a true regulator of MSLN. Moreover, in vitro experiments showed that treatment with miR-21-5p mimic reduced proliferation of MPM cell lines. Altogether, this work shows that the miR-CATCH technique, coupled with NGS and in vitro validation, represents a reliable method to identify native miRNA::mRNA interactions. MiR-21-5p is suggested as novel regulator of MSLN with a possible functional role in cellular growth.

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Background Hip fracture is a growing healthcare challenge, with 6-8% 30-day mortality and 20-30% of patients incurring major morbidity, including impaired mobilisation and ability to live independently. While observational studies have shown no benefit of general versus spinal anaesthesia on 30-day mortality, intraoperative hypotension during hip fracture surgery is associated with increased 30-day mortality regardless of anaesthetic technique. Although a recent trial on younger patients demonstrated reduced postoperative complications by maintaining intraoperative arterial blood pressure close to preoperative baseline, there are no data correlating intraoperative hypotension during hip fracture surgery with postoperative morbidity. Objective We evaluated the hypothesis that duration and severity of intraoperative hypotension during hip fracture surgery is associated with increased postoperative morbidity. Methods A retrospective analysis was carried out on n = 52 patients undergoing hip fracture surgery between January and June 2017. Measurements of patients' intraoperative systolic arterial pressure (SAP) and mean arterial pressure (MAP) during anaesthesia, logged electronically through an anaesthesia information management system, were reviewed. We calculated the total duration of time where SAP or MAP were below pre-defined thresholds for hypotension (MAP < 75 mmHg, MAP < 55 mmHg, SAP ≤ 80% admission baseline or SAP ≤ 80% pre-induction baseline). Univariate and bivariate descriptive statistics were generated for all relevant variables. With multivariable regression models containing known predictors, cumulative duration of hypotension was correlated with postoperative comorbidities as quantified by the Clavien-Dindo and Comprehensive Complication Indices. Results Mean age (± SD) was 78 ± 13 years, 75% were female, 87% were ASA II or III and 60% underwent spinal anaesthesia. Mean Comprehensive Complication Index was 20.4 ± 19.2. Lowest absolute SAP and MAP values were 82 ± 18 mmHg and 55 ± 12 mmHg respectively. Cumulative time of SAP < 80% pre-induction value adjusted to gender, age and the Charlson Comorbidity Index was associated with progression to a higher Clavien-Dindo classification (odds ratio 1.020 per additional minute; 95% CI 1.008-1.035; P = 0.003). Conclusions In this exploratory retrospective analysis, the cumulative time of hypotension during hip fracture surgery correlated with extensive postoperative morbidity when adjusting to other known predictors. Intraoperative cumulative time of hypotension may be a good candidate for larger prediction studies as a predictor of postoperative complications. A randomised controlled trial evaluating the effect of actively minimising intraoperative hypotension on postoperative morbidity in hip fracture patients seems warranted.

Background Classical galactosemia (CG) (OMIM #230400) is a rare disorder of carbohydrate metabolism, due to deficiency of galactose‐1‐phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long‐term complications, mainly cognitive, neurological, and female infertility remains poorly understood. Objectives This study investigated (a) the association between specific IgG N‐glycosylation biomarkers (glycan peaks and grouped traits) and CG patients (n = 95) identified from the GalNet Network, using hydrophilic interaction ultraperformance liquid chromatography and (b) a further analysis of a GALT c.563A‐G/p.Gln188Arg homozygous cohort (n = 49) with correlation with glycan features with patient Full Scale Intelligence Quotient (FSIQ), and (c) with galactose intake. Results A very significant decrease in galactosylation and sialylation and an increase in core fucosylation was noted in CG patients vs controls (P < .005). Bisected glycans were decreased in the severe GALT c.563A‐G/p.Gln188Arg homozygous cohort (n = 49) (P < .05). Logistic regression models incorporating IgG glycan traits distinguished CG patients from controls. Incremental dietary galactose intake correlated positively with FSIQ for the p.Gln188Arg homozygous CG cohort (P < .005) for a dietary galactose intake of 500 to 1000 mg/d. Significant improvements in profiles with increased galactose intake were noted for monosialylated, monogalactosylated, and monoantennary glycans. Conclusion These results suggest that N‐glycosylation abnormalities persist in CG patients on dietary galactose restriction which may be modifiable to a degree by dietary galactose intake.

<b><i>Background:</i></b> Sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor licensed for the treatment of type 2 diabetes mellitus (T2DM), has been reported to improve psoriasis. <b><i>Objective:</i></b> We compared the effects of sitagliptin treatment, a DPP-4 inhibitor, in combination with narrow-band ultraviolet-B (NB-UVB) phototherapy compared to NB-UVB alone on psoriasis severity, quality of life, cardiovascular disease risk factors and immune parameters in people with moderate psoriasis without T2DM. <b><i>Methods:</i></b> In this 39-week, single-centre, randomised controlled trial, people were allocated randomly to receive sitagliptin for 24 weeks with NB-UVB or NB-UVB alone. The primary endpoint was the change in Psoriasis Area and Severity Index (PASI) from baseline to 24 weeks. We estimated that 120 participants would be needed to have 80% power to find a significant difference between the groups. <b><i>Results:</i></b> A total of 118 patients were randomised. The median (IQR) baseline PASI was 8.8 (7.5–11.6). At 24 weeks, the mean difference from baseline in PASI (–1.0 [95% CI –2.0 to 0.0]) was significantly larger in the sitagliptin/NB-UVB arm than in the NB-UVB-alone arm (<i>p</i> = 0.044). There were significant differences in the change in Hospital Anxiety and Depression Scale (–2.5 [95% CI –4.0 to –1.0]; <i>p</i> = 0.002) and EuroQol 5-item questionnaire (0.1 [95% CI 0.0–0.1]; <i>p</i> = 0.036) values from baseline to 24 weeks between the sitagliptin/NB-UVB and the NB-UVB-alone arm. There were no treatment-related serious adverse events. <b><i>Conclusion:</i></b> Sitagliptin therapy combined with NB-UVB phototherapy significantly improved psoriasis severity, albeit modestly, compared to NB-UVB phototherapy alone in patients with moderate psoriasis without T2DM.

Summary Intrathecal morphine is an analgesic option for major hepatopancreaticobiliary procedures but is associated with a risk of respiratory depression. We hypothesised that a postoperative low‐dose naloxone infusion would reduce the incidence of respiratory depression without an increase in pain scores. Patients scheduled for major open hepatopancreaticobiliary surgery and who were receiving 10 μg.kg−1 intrathecal morphine were eligible for inclusion. Patients were allocated randomly to receive a postoperative infusion of naloxone 5 μg.kg−1.h−1 (naloxone group) or saline at an identical infusion rate (control group) until the morning after surgery. Clinicians, nursing staff and patients were blinded to group allocation. The primary outcome measure was the incidence of respiratory depression (respiratory rate < 10 breaths.min−1 and/or oxygen saturation < 90%). Secondary outcome measures included: arterial partial pressure of carbon dioxide; pain score; requirement for supplemental analgesic; and incidence of nausea and vomiting, pruritus and sedation. In total, data from 95 patients (48 in the naloxone group and 47 in the control group) were analysed. The incidence of respiratory depression was lower in the naloxone group compared with the control group (10/48 vs. 21/47 patients, respectively; p = 0.037, relative risk 0.47 (95%CI 0.25–0.87). Maximum pain scores were greater for patients allocated to the naloxone group compared with control (median 5 (95%CI 4–6) vs. 4 (95%CI 2–4), respectively; p < 0.001). A low‐dose naloxone infusion decreases the incidence of respiratory depression following intrathecal morphine administration in patients having major hepatopancreaticobiliary surgery at the expense of a small increase in postoperative pain.

Genetic engineering of mammalian cells is of interest as a means to boost bio-therapeutic protein yield. X-linked inhibitor of apoptosis (XIAP) overexpression has previously been shown to enhance CHO cell growth and prolong culture longevity while additionally boosting productivity. The authors confirmed this across a range of recombinant products (SEAP, EPO, and IgG). However, stable overexpression of an engineering transgene competes for the cells translational machinery potentially compromising product titre. MicroRNAs are attractive genetic engineering candidates given their non-coding nature and ability to regulate multiple genes simultaneously, thereby relieving the translational burden associated with stable overexpression of a protein-encoding gene. The large number of potential targets of a single miRNA, falsely predicted in silico, presents difficulties in identifying those that could be useful engineering tools. The authors explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH protocol. A biotin-tagged antisense DNA oligonucleotide for XIAP mRNA is designed and used to pull down and capture bound miRNAs. Two miRNAs are chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both results in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increases XIAP protein levels. A 3'UTR reporter assay confirms miR-124-3p to be a bona fide regulator of XIAP in CHO-K1 cells. This method demonstrates a useful approach to finding miRNA candidates for CHO cell engineering without competing for the cellular translational machinery.